Detach HEK cells using Accutase for 5 minutes at 37°C and collect them.

Spin cells in a centrifuge at 250g for 5 minutes at room temperature.

Remove the supernatant and wash cells in PBS.

Repeat steps 2 and 3 for a total of 2 washes.

Lyse cells in 1% TBS + 0.5% NP40 + PI/PHI (Pierce #A32959)

Filter lysates through a 0.22uM PES syringe filter (Millipore).

Wash 50ul of M2 antiflag agarose resin (Sigma–Aldrich, #A2220) according to the manufacturer's instructions for 10 mg of total protein.

Incubate 10 mg of lysates with prewashed M2 anti-flag agarose resin for 2h at 4°C on a rotating wheel.

Elute Flag with Flag Peptide (Sigma-Aldrich, #F3290) by moving on a wheel for 20 min at 4°C followed by a spin for 1 min at 1000 g.

Collect the flow-through and proceed with TMT labeling or Western Blot analysis.